Optimization and validation of a micro–QuEChERS method for phthalates detection in small samples of cetacean blubber

In this study, an innovative method was developed to detect and quantify phthalates in fresh cetacean blubber. An adaptation of the ammonium formate QuEChERS method was used and adapted as a micro-extraction for small quantities of samples. Significantly, this technique utilized minimal quantities of reagents and salts, with the additional implementation of rigorous Quality Assurance/Quality Control protocols to further reduce background contamination. To ensure the reliability of this method, comprehensive validation procedures were conducted, with a specific focus on two widely studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus). Determination coefficients (R2) for matrix-matched calibration were >0.93 with limits of quantifications (LOQ) of the method in the range of 5–10 ng/g. Mean recovery values were between 40 and 100 %. This novel methodology holds particular relevance for environmental research studies, offering the capability to detect emerging contaminants with minimal sample requirements. This aspect is particularly valuable in investigations that involve free-ranging animals and rely on biopsy sampling. It allows for the assessment of contaminant levels in healthy individuals within wild populations, enhancing our understanding of ecological impacts and potential conservation measures.• A micro-extraction adaptation of the ammonium formate QuEChERS method was developed and applied to a small quantity of fresh cetacean blubber to detect phthalates.• Small quantities of reagents and salts were used, and additional Quality Assurance/ Quality Control procedures were taken to further minimize background contamination.• Method validation was carried out for two cosmopolitan and extensively studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus).

• A micro-extraction adaptation of the ammonium formate QuEChERS method was developed and applied to a small quantity of fresh cetacean blubber to detect phthalates.• Small quantities of reagents and salts were used, and additional Quality Assurance/ Quality Control procedures were taken to further minimize background contamination.• Method validation was carried out for two cosmopolitan and extensively studied cetacean species: the common bottlenose dolphin ( Tursiops truncatus ) and the short-finned pilot whale ( Globicephala macrorhynchus ).

Method details
A modified version of the QuEChERS method described in Sambolino et al. [1] was applied to a small quantity of cetacean blubber for phthalates extraction and purification (named micro-QuEChERS).The present method, in order to be applied in 100x smaller quantity of tissue, sees a considerably reduced amount of extraction reagents, while maintaining the same amount of purification salts, given the higher fat concentrations of the target tissue.An extra step was introduced to concentrate the final extract, given the limited amount of analytes extractable from such small samples.For this reason, additional cleaning procedures were implemented to control background contamination, minimizing the risk of co-extraction of phthalates in reagents and glassware.The target analytes in this modified approach are seven phthalates which are the most commonly encountered in the marine environment [2][3][4] .
Blubber tissue was collected from stranded cetaceans (common bottlenose dolphin -Tursiops truncatus and short-finned pilot whale -Globicephala macrorhynchus ) and kept frozen at − 20°C until the day of analysis.On the analysis day, a small portion of sample (50 mg, wet weight [w.w.]) was cut, added to a 15 mL glass tube, and mashed, still frozen, with a glass rod for at least 1 min.One ml of Acetonitrile (ACN) was then added to the glass tube with the freeze-homogenized sample, vortexed for 1 min, then 0.5 g of ammonium formate was added, in order to induce phase separation [5] ; the mixture was vortexed again for 1 min and then centrifuged for 5 min at 2500 rpm.For the purification step, all the supernatant was transferred to a glass tube containing 150 mg of MgSO 4 , 50 mg of Primary Secondary Amine (PSA), and 50 mg of C18.The ratio of ammonium formate to ACN and MgSO 4 in PSA and C18 followed established methodologies [ 1 , 5 ], although quantities of reagents were considerably reduced in comparison to the original method [1] .The mixture was again vortexed for 1 min and centrifuged for 5 min at 2500 rpm.The resulting supernatant (200 μl) was transferred to a GC vial, evaporated overnight, and then reconstituted in 50 μl of Cyclohexane (CH).
To minimize contamination, all the glassware was carefully cleaned, immersed in an acid bath for 24 h, and then muffled at 550°C overnight [ 1 , 6 ].No plastic material besides pipette tips (phthalate-free) was used during the procedure.To minimize background contamination from organic reagents, ACN and CH were double distilled before use [7] and salts were washed three times with methanol.Two procedural blanks were analyzed with each batch of samples, and an average of the two blank values (RSD < 10 %) was subtracted from the final results.High-purity solvents and reagents were used.Methanol, ACN and CH of LC-MS grade, ammonium formate (purity ≥ 97.0 %) and MgSO 4 (purity ≥ 98.0 %) were from VWR International Eurolab (Barcelona, Spain).PSA and C18 were from Agilent Technologies (Santa Clara, CA, USA).
Seven phthalates (Phthalic Acid Ester -PAE) were the target analytes (chemical structures and proprieties in Table 1 ).High purity standards ( > 98 %) of each PAE and two isotopically labeled PAEs (DEP-D4 and DBP-D4, used as internal standards) were acquired from Sigma-Aldrich (Madrid, Spain) and Dr. Ehrenstorfer (Augsburg, Germany).The determination and quantification of the analytes was carried out with an Agilent 6890 GC (Agilent Technologies, California, USA), equipped with an Ultra Inert HP-5 ms capillary column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness), coupled with an Agilent 5973 Network MS (Agilent Technologies, California, USA), in the selected ion monitoring (SIM) mode.The oven temperature program started at 60 °C (1 min), then increased to 190 °C at 45 °C/min, and finally increased to 310 °C at 35 °C/min and held for 3 min.Injection was in splitless mode (purge time 0.75 min; purge flow 40 ml/min) at 280 °C, with an injection volume of 2 μl.The detection parameters follow those described in Sambolino et al. [1] ; retention times, ion qualifiers and quantifiers of selected PAEs for this study are provided in Table 2 .

Method validation
The modified QuEChERS method (micro-QuEChERS) was validated through several steps.First, an instrument calibration using the internal standard approach was done in order to quantify the blank values and calculate the matrix effect ( Table 3 ).Recoveries studies at two spiking levels (25 and 150 ng/g w.w.) and matrix-matched calibrations with the internal standard method were then carried out for both matrices (blubber samples from the two species) ( Tables 4 and 5 ).The equations coefficient obtained were used to quantify the compounds in the analyzed samples.The studied linear range, the matrix effect (ME) and the limits of quantifications (LOQ) of the method, considered as the lowest calibration level with signal to noise ratio > 10, are reported in Table 5 for the two different matrices analyzed.
The method yielded recoveries with relatively high variation.For T. truncatus samples, mean relative recoveries were between 85-100% for five of the seven PAEs; DMP and DNOP held lower recoveries (40 % and 75 %, respectively).For G. macrorhynchus , mean recoveries ranged between 53 % and 100% ( Table 4 ).Matrix-matched calibrations with the internal standard method were calculated for each matrix, obtaining linear regression with fitting R 2 > 0.93.Limits of quantifications (LOQ) of the method ranged between 5 and 10 ng/g w.w. ( Table 5 ).The matrix effect varied depending on the matrix and the analyte, staying within the ± Level 1: 25 ng/g of w.w; level 2: 150 ng/g of w.w.Data outside the 70-120 % range for recovery values and 0-20 % for RSD values are in bold.

Additional information
The present manuscript proposes a new rapid, cost-effective methodology to analyze phthalates in small blubber samples from two odontocete species.Bottlenose dolphins and pilot whales, extensively studied among delphinid species due to their widespread abundance and distribution in temperate and tropical waters [ 8 , 9 ], face significant anthropogenic impacts, sharing habitats and resources with human activities [ 1 , 10 ].Conservation efforts necessitate monitoring their contaminant status.The use of biopsy

Table 6
Results of the phthalates (PAEs) analysis of cetacean blubber samples from biopsies of short-finned pilot whales ( Globicephala macrorhynchus, n = 15) and common bottlenose dolphins ( Tursiops truncatus, n = 9).< LOQ: below the limits of quantification, 1 2 of the LOQ value was considered for statistical analysis; n.d.: analyte not detected (below limit of detection); values in bold have RSD > 20 %. sampling in free-ranging individuals offers a superior assessment of wild population health compared to stranded animals [ 11 , 12 ].However, these samples are extremely limited in quantity, demanding innovative methodologies for their effective use.To the best of our knowledge, only one other method [13] has been developed for working with such samples.Our refined approach, aligning with QuEChERS extraction principles, proves notably more efficient and faster, utilizing only half the sample quantity (50 mg vs. 100 mg).

Matrix (species)
Previous studies [ 14 , 15 ] indicate that pollutant concentrations may vary based on blubber layer stratification and sample body location.Therefore, analyzed biopsy samples should encompass all layers, from beneath the skin to above the muscle, with due consideration to the sample's body location.
The proposed method is anticipated to produce comparable results for other odontocete species, however, blubber tissue in different cetacean species exhibits variability in thickness and triglycerides/wax esters composition [16] .Thus, when applying the method to new species, validation is strongly recommended.
Analyzing blubber is challenging due to its high lipid content, posing interference with chromatographic analysis.While employing ACN facilitates lipid removal from fat tissues, an additional cleanup step is essential [17] .Dispersive solid-phase extraction (d-SPE) with a mixture of MgSO4, PSA and C18 was found effective for coextractives removal from acetonitrile extract [18] .However, some coeluates extracted during the procedure may interfere with the final analysis, causing signal suppression [19] , which was observed in the matrix effect ( Table 5 ).Recovery rates for most analytes ranged from 80 to 100 %.However, analytes like DMP exhibited a significant loss, potentially attributed to the drying step, resulting in a lower rate of 40 %.

Declaration of generative AI and AI-assisted technologies in the writing process
During the preparation of this work the authors used ChatGPT in order to improve text fluency and readability.After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Table 1
Chemical structure and properties of the studied phthalates.
a 24 °C.Data taken from SciFinder® and PubChem databases.MM: Molecular mass.

Table 2
Retention longitudinally cut to encompass all the blubber layers.Results on the PAEs concentrations are reported in Table6and are further discussed in the related research article.

Table 3
Internal instrumental calibration data of the target analytes.

Table 4
Relative recovery (%) and RSD values (in brackets) of the target analytes from recovery studies with two spiking levels on blubber samples.